家蝇触角cDNA文库的构建及质量分析  

Construction of a cDNA Library of the Antenna of Housefly, Musca domestica

作　　者：朱彬彬[1] 姜勇[1,2] 牛长缨[1,2] 张长禹 雷朝亮[1,2] 

机构地区：[1]华中农业大学昆虫资源研究所,湖北武汉430070 [2]宜昌市疾病预防控制中心,湖北宜昌443000

出　　处：《Zoological Research》2005年第2期203-208,共6页动物学研究（英文）

基　　金：湖北省自然科学基金项目(2001ABB128)

摘　　要：采用SMART(switchingmechanismat5′endofRNAtranscript)技术,构建了家蝇(Muscadomestica)的cDNA质粒文库。按TRIzolReagent试剂盒的操作说明提取家蝇触角总RNA,以此为模板,通过PowerScriptTM反转录酶反转录合成第一链cDNA;再以第一链cDNA产物为模板,用LD PCR合成cDNA双链。该cDNA产物经分级分离、连接和电转化,即得到家蝇触角cDNA原始文库,其滴度为6.3×108cfu/mL。对原始文库进行扩增,取适量扩增文库稀释并涂平板,用PCR方法测得文库的重组率为91%,插入cDNA的平均长度为1.7kb。随机挑取克隆提取质粒,5′端测定插入片段的核苷酸序列,克隆得到一个具有5′和3′非编码区的气味结合蛋白OBP3基因(MdomOBP3,GenBank登录号AY826189)。测序分析表明,该基因具有气味结合蛋白的标志性结构域;由推导的氨基酸序列同源性分析表明,该基因与已报道的双翅目昆虫气味结合蛋白OBP有较高的同源性。In order to understand the molecular mechanism of olfaction of housefly, Musca domestica, we constructed a cDNA library using the Creator^TM SMART^TM cDNA library construction kit (Clontech). Total RNA was isolated from the housefly antenna using TRIzol Reagent. The PowerScript^TM reverse transcriptase was used to synthesize and anchor the first-strand cDNA. Following reverse transcription, LD-PCR was performed using a modified oligo (dT) and an anchor primer to enrich the cDNA population for full-length sequences. cDNA size fraction, ligation and transformation into E. coli were continued to generate the oriental, unamplified cDNA library. The unamplified cDNA library has a titer of about 6.3×108 cfu/mL, in which the recombinant clones with an average insert size of 1.7 kb were about 91%. Amplification of oriental library is vital to obtain enough high quality plasmid for library screening and for long-term storage of the library. A full-length odorant binding protein (OBP) cDNA with 5′ and 3′ untranslated regions was isolated from the cDNA library, and named MdomOBP3 (GenBank Accession: AY826189). Sequencing and analysis show that the OBP cDNA is characterized by typical conservative Cys. Deduced amino acid sequence is highly similar to that of six OBPs from Diptera, with sequence identity of 59%-82%.

关 键 词：家蝇 触角 CDNA文库 SMART 气味结合蛋白 OBP3基因 嗅觉 

分 类 号：Q963[生物学—昆虫学]